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Year : 2020  |  Volume : 4  |  Issue : 1  |  Page : 76-80

In vitro antiproliferative activity of cold atmospheric plasma on small-cell lung carcinoma

1 Department of Physics, Shahid Beheshti University, Tehran, Iran
2 Mycobacteriology Research Centre, National Research Institute of Tuberculosis and Lung Disease, Shahid Beheshti University of Medical Sciences, Tehran, Iran
3 Mycobacteriology Research Centre, National Research Institute of Tuberculosis and Lung Disease; Department of Biotechnology, School of Advanced Technologies in Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran
4 Laser and Plasma Research Institute, Shahid Beheshti University, Tehran, Iran

Date of Submission03-Jan-2020
Date of Acceptance03-Feb-2020
Date of Web Publication17-Mar-2020

Correspondence Address:
Dr. Jalaledin Ghanavi
Mycobacteriology Research Centre, National Research Institute of Tuberculosis and Lung Disease, Shahid Beheshti University of Medical Sciences, Tehran
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/bbrj.bbrj_25_20

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Background: Cold plasma is shown to inhibit the cancer cell growth. Manipulation of different plasma parameters might have influence on the production of major reactive species which leads to killing of the cancer cells. Antiproliferative activity of cold atmospheric pressure plasma jet was investigated on small-cell lung carcinoma BHY cell line (squamous cell carcinoma) under different in vitro conditions. Methods: A homemade plasma jet was designed and created using pure helium gas. To identify the species created by the plasma jet, optical emission spectroscopy (OES) was employed. Next, the effect of plasma jet was examined on lung cancer cell survival by MTT assay and the effects of main parameters were evaluated on plasma performance. In this favor, various treatment times including 60, 90, 120, 180, and 300 s in combination with different voltages of 5, 11, and 14 kV were investigated, and the results were analyzed at 2, 24, and 48 h after exposure to plasma. Results: Predominant species of OES spectra were O, OH, N2+, and N2. Results of MTT assay indicated a dramatic reduction in cell viabilities in both dose- and time-dependent manners, and more than 75% of cancer cells were died after 48 h at 180 s of plasma treatment. Conclusion: The homemade plasma jet can chiefly contribute to the production of reactive oxygen and nitrogen species (reactive oxygen species and reactive nitrogen species) and can induce apoptosis in small-cell lung carcinoma BHY cell line.

Keywords: Anti-tumor therapy, cold atmospheric plasma, plasma jet, small-cell lung carcinoma

How to cite this article:
Amini M, Ghanavi J, Farnia P, Karimi M, Ghomi H. In vitro antiproliferative activity of cold atmospheric plasma on small-cell lung carcinoma. Biomed Biotechnol Res J 2020;4:76-80

How to cite this URL:
Amini M, Ghanavi J, Farnia P, Karimi M, Ghomi H. In vitro antiproliferative activity of cold atmospheric plasma on small-cell lung carcinoma. Biomed Biotechnol Res J [serial online] 2020 [cited 2022 Oct 7];4:76-80. Available from: https://www.bmbtrj.org/text.asp?2020/4/1/76/280878

  Introduction Top

Lung cancer is one of the most common causes of cancer death worldwide and is currently considered as an epidemic on a global scale.[1] This malignancy was responsible for 29% of all deaths from cancer (31% of men and 26% of women).[2]

There are several ways to manage lung cancer treatment, including surgery, radiation therapy, and chemotherapy.[3] However, due to its malignancy and aggressiveness, the treatments often fail to control the disease. Being one of the most common leading causes of cancer death, it garners much interest by researchers to develop innovative treatments. Plasma jet is a novel technique which is recently being introduced for cancer therapy. Plasma is the fourth state of matter that is well-known as partially or completely ionized gas such as a mixture of electrons, ions, radicals, and high-energy photons, which are usually produced under high-temperature conditions.[2] Today, cold atmospheric nonthermal plasma, a type of plasma that is formed at relatively “cold” temperatures or room temperature, has gained tremendous attention in biomedical engineering.[4] Plasma sources operating at body temperature and under atmospheric pressure provide the opportunity for their medical uses [5] and with modern applications of cold plasma types, a fruitful arena for cancer therapy is now being emerged. Several evidences suggest that plasma treatment can have different effects on multiple cancer cell types, reactive oxygen species (ROS) and reactive nitrogen species (RNS) during oxidative stress, mitochondrial interface-dependent apoptosis,[6],[7] cell cycle arrest, cell growth inhibition, and blocking cell invasion.[7],[8]

In this study, the effect of atmospheric pressure cold plasma was investigated on human lung cancer cell line, BHY (squamous cell lung carcinoma). Viabilities of the cells were evaluated using MTT assay and cell apoptosis was assessed using APPOP ladder kit [Figure 1]. Plasma treatment was carried out using plasma jet equipment with pulsed DC power supply at fixed pure helium gas flow rate of 1.5 L/min for different treatment times and voltages. This study was designed to investigate the probable role of cold atmospheric plasma therapy in lung cancer treatment and the conditions of its usage.
Figure 1: Picture of the experiment: Performed in our laboratory (a) schematic of the experimental setup; (b) plasma jet over the sample

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  Methods Top

Ethical Issue

The study protocol was approved by the Research Ethics Committee of the National Research Institute of Tuberculosis and Lung Disease, Shahid Beheshti University of Medical Sciences, Tehran, Iran (IR.SBMU.NRITLD.1395.255).

Experimental setup of plasma jet

A 6 kHz pulsed DC power supply was used to ignite the plasma jet under different voltages with a maximum of 14 kV. Helium was used as the working gas for this plasma jet at flow rate of 1.5 L/min which flowed through the quartz capillary (internal diameter 4 mm, external diameter 6 mm, and length 13 cm). The high power electrode was a copper wire which was placed at the outside of the capillary, 1 cm away from the end of it. The capillary and the power electrode were housed in a Teflon tube. The plasma jet was ignited easily by applying the power supply and a stable plasma jet was achieved at room temperature. [Figure 1]a represents a typical schematic of the experimental setup when the instrument is running.

Optical emission spectroscopy

Optical emission spectroscopy (OES) is one of the most common methods for the analysis of the plasma.[9] In the present study, a range of wavelengths from 200 nm to 900 nm, i.e., UV-VIS-NIR, was applied with an OES (SV 2100, K-MAC) to identify the species in the plasma created by cold atmospheric pressure helium jet (atomic oxygen [O], hydroxyl radical [-OH], nitrogen [N2], nitrogen cation [N2+], and atomic hydrogen [H]).

Cell culture

The human squamous cell lung carcinoma BHY was purchased from the Pasteur Institute of Iran, Tehran, Iran. Subsequently, the cell line was cultured in Dulbecco's Modified Eagle's Medium (DMEM/F-12 [1:1]) supplemented with 10% (v/v) fetal bovine serum plus penicillin (100 unit/ml) and streptomycin (100 μg/ml), followed by incubation at 37°C in 90% humidity and 5% (v/v) CO2. Cells were observed under a Nikon Eclipse TS100 inverted microscope.[10]

Plasma treatment

To probe the effect of plasma treatment on cancer cells, 1 day before plasma irradiation, 104 cells per well were cultured in 96-well plate. The treatment conditions were as follows:[11] the plate-to-nozzle separation distance was set at 3.5 cm and pure helium gas was selected at flow rate of 1.5 L/min. The first well was considered as the control and hence, it was not irradiated with plasma. Various exposure times, including 60, 90, 120, 180, and 300 s in combination with different voltages of 5, 11, and 14 kV were examined and the results were analyzed at 2, 24, and 48 h after exposure to plasma.[11] Each experiment was carried out in triplicate. [Figure 1]b shows the plate when it is located at a distance of ~ 3.5 cm from the outlet of the plasma jet device.

Cell viability assay

The antiproliferative effects were measured by MTT assay which is a quantitative colorimetric method for measuring the activity of mitochondrial dehydrogenase enzymes in the cell that reduce MTT reagent to its insoluble formazan crystals, turning to a purple color [Figure 1]. In fact, mitochondrial dehydrogenase enzymes in living cells replace the bromine in the solution with hydrogen and as a result, MTT is converted to formazan.[12] In this regard, 96-well plate-containing treated cells was transferred from incubator under the hood and 20 μl of yellow MTT solution was added to each well, followed by incubation for another 1.5 h. Plates were then brought out from the incubator, and the cells were observed under the microscope; the living cells were appeared in blue because of the absorption of formazan crystals. Next, the supernatants were removed by pipette and 100 μl of DMSO solution was added to each well as the solvent. Eventually, the absorbance of the cells was read at 490 and 650 nm under ambient temperature using a microplate reader Bio Tek 800.[12]

Determination of cell apoptosis

The induction of programmed cell death was determined morphologically after incubation [Figure 1]. For a more detailed pattern, after 2, 24, and 48 h of treatment with plasma irritation, DNA cells were extracted using APPOP Ladder kit and DNA fragments were subsequently visualized by agarose gel electrophoresis.[13]

Data analysis method

Statistical analysis of all experiments related to MTT assays was performed in three biological replicates. All quantitative data were analyzed using SPSS software version 22 (IBM Corporation, Armonk, NY 10504, U.S.A.); in a way that all data were evaluated for normal distribution using Shapiro–Wilk test. MTT data displayed normal distribution and were evaluated using one-way ANOVA method, and then were compared in two-by-two groups using the least significant difference test. Data were presented in mean ± standard deviation (SD) for each test, and P < 0.05 was considered statistically significant. The resulting graphs were plotted based on mean ± SD using GraphPad Prism.

  Results Top

Optical emission spectroscopy

Optical emission spectra were analyzed to identify cold atmospheric pressure plasma jet particles and radicals, which may account for the observed effects of the plasma on cancer cells.[14] [Figure 2] represents the OES spectrum of the helium cold plasma jet, interacting with the ambient air at helium flow rate of 1.5 L/min. As it is evident from this figure, plasma jet can play an important role in the production of RNS and ROS.
Figure 2: Optical emission spectrum of cold atmospheric pressure plasma jet during discharge; determination of the major reactive species is shown

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The emission lines and bands were mainly identified according to the reference.[15] Dominant emission lines indicate the presence of helium ions (He) at 667–706 nm and atomic oxygen (O) at 777 nm. Atomic oxygen (O) (including the ground state as well as all excited states of the atomic oxygen) is believed to significantly influence the cells and hence has a broad range of biomedical applications. In addition to the species identified with nitrogen, nitrogen molecules (N2) ranging from 300 to 350 nm and the ionized nitrogen molecules (N2+) in the range of 380–427 nm were also present. Repeated measurements were carried out to confirm the results. Previous studies have indicated that the inactivation effect of ultraviolet (UV)-radiation on bacteria is mainly attributed to DNA/RNA damage at UV wavelengths of 200–280 nm.[16] Accordingly, it can be concluded that UV photons are not major species of the cold atmospheric pressure plasma jet in the present work. Cold plasma jet is a complicated environment which combines the comprehensive effect of different ions and neutral species. As it is apparent from [Figure 2], predominant species of the spectra are O (777 nm), OH (309 nm), N2+ (380, 427 nm), and N2(340 nm).

Voltage variation effect on reactive oxygen species(ROS) production

Increasing the voltages from 5 to 14 kV resulted in a rise in corresponding intensities of each species [Figure 2]. The increasing trend of major ROS and RNS species is illustrated in [Figure 3]a. Helium line at 706 nm is believed to provide energetic electrons,[17] and recent studies have shown that it has been linked to energetic electrons in RF atmospheric plasmas.[18] Therefore, intensities of the major ROS and RNS species were normalized to He line at 706 nm [Figure 3]b. The flat lines indicate that the rise for each species is proportional to the voltage increasing from 5 to 14 kV.
Figure 3: Effect of increasing the voltage from 5 to 14 kV on intensity of each species: (a) Upward trend of major reactive oxygen species and reactive nitrogen species by increasing the voltage. (b) The rise in each species is proportional to the voltage increasing from 5 to 14 kV when normalized to He (706 nm)

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Analysis of MTT assay and Appop ladder kit in plasma-treated cells

In MTT assay, living cells absorb the yellow soluble MTT reagent and produce blue insoluble formazan, forming microscopic crystals in cell culture. Colorless cells represent dead cells. [Figure 4] depicts the cells after 1.5 h of MTT addition. As is evident, plasma treatment has reduced the viability of the cells. Electrophoresis of DNA extracted from BHY cells after treatment with different voltages and incubation times was investigated to confirm the fragmentation of DNA bands and approve the apoptosis.
Figure 4: Morphological changes under light microscopy: cell shrinkage and chromatin condensation>

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Next, to investigate the effect of exposure time, percentages of cell viabilities were determined for cancer cells treated with plasma for various time durations from 5 to 300 s under different voltages. Results indicated that plasma treatment dramatically reduced the viability rate of human lung carcinoma cells in both dose- and time-dependent manners [Figure 5]. In general, a rise in the applied voltages would result in a corresponding decline in cell viabilities. The production of reactive species with higher intensities under higher voltages may account for this [Figure 3]. A downward trend was also observed in cell viabilities over the 60–300 s for all the applied voltages. Results from the cells at different times of 2, 24, and 48 h after treatment demonstrated that plasma anticancer effects were significantly more pronounced at 48 h in comparison with 2 and 24 h. Previous studies have indicated that ROS and RNS can produce new powerful reactive species (e.g., peroxynitrite, ONOO ) once they collide or come into contact with each other over long periods.[19] While the cells continuously remove the primary oxygen and nitrogen species to reduce their harmful effects, fail to neutralize newly formed reactive species,[20] which may result in substantial oxidation and potential disruption of cell signaling pathways, leading to cell death.[21] This may explain why cell viabilities did not decline much during 2 and 24 h after plasma treatment but dropped drastically after 48 h [Figure 5].
Figure 5: MTT assay results of BHY cells treated with 5.300 s duration of helium plasma jet using different voltages at (a) 2 h, (b) 24 h, (c) 48 h. Diagram on the percentage of viability of the cells after 2 h (5, 11, and 14 kV in the first, second, and third rows, respectively). Data are shown as mean } standard deviation; (n = 3); *P < 0.01, **P < 0.001

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Overall, the results revealed that after 48 h, approximately more than 75% of cancer cells died at 180 and 300 s of plasma treatment (P values showed statistically significant differences compared to the control). Therefore, it might be recommended that the threshold needed for plasma therapy of BHY cells is approximately 180 s. Results also displayed that exposure times of 5–15 s caused an increase in the number of cells, and hence, it can be concluded that the time duration of plasma therapy for BHY cells should be more than 15 s.

  Discussion Top

In recent years, many efforts have been made to provide new methods for cancer treatment, among which the choice of cold atmospheric plasma therapy can be of great interest to researchers because of its proper functioning and selectiveness (does not exert harmful effects on normal tissues). Previous studies have indicated that plasma can inhibit the growth of cancer cells. It has been shown that plasma needle leads to cell detachment in cancer cells [22] and floating electrode dielectric barrier discharges in the air promote apoptotic behavior in melanoma skin cancer cell lines.[23]

Although its exact mechanism of action is not clearly understood, many researchers have attributed the performance of plasma to its ingredients such as hydrogen peroxide, hydroxyl, and alkoxyl radicals as well as ROS and highlighted their roles in plasma function.[24] Each species may cause significant damage to cell structures, membrane or DNA in such a way that leads to cancer cell killing.[25] One example is the occurrence of oxidative stress as a result of the excessive production of free radicals and ROS, which can be reduced by antioxidant factors. In fact, an imbalance between the production of free radicals and peroxides and responses of the antioxidant defense system leads to oxidative stress.[26] The presence of low concentrations of ROS in transducing intracellular messages has been proved during cell lysis.[27] Recently, an interesting mechanism for the antitumor function of reactive species derived from cold atmospheric plasma has been hypothesized.[28] The cold atmospheric plasma-derived 1 O2 can inactivate membrane-bound catalase whose presence is required for tumor progression; thus, inducing RONS-dependent apoptosis selectively in tumor cells.

In the present study, comparison of the results of irradiation on cells at different times of 2, 24, and 48 h revealed that the minimum percentage of survival was observed in cells treated with 11 and 14 kV plasma radiations and exposure time of 48 h after treatment. Thereby, more cells are dead, that is, by applying higher voltages of plasma jet and increasing the time of irradiation, the more cells undergo apoptosis. The increase in treatment time and the voltage causes the rise in the density of reactive species produced in plasma, which subsequently increases apoptosis. Accordingly, the decline in metabolic activity of the cells treated with plasma can be associated with the creation of ROS and RNS, which eventually leads to a reduction in cancer cell growth.

Atmospheric plasma acts at room temperature in the open air, and hence, it may offer the remarkable potential for therapeutic activity in the treatment of cancer. In particular, by manipulating different plasma parameters, cancer cells might be selected from healthy ones that is considered as great merit in postoperative treatments.

  Conclusion Top

Two of the most important parameters in plasma treatment are the time duration of exposure to plasma and the output voltage difference at the electrodes. These factors individually and in combination with each other, resulting in a respective decline in cell viability of BHY cells. The mechanism of plasma therapy for the removal of cancer cells was estimated by measurement of RNS and ROS under different voltages. The relationship between these factors and the production of reactive species was investigated by spectral measurements and MTT assay which showed that the voltage plays a role in creating larger concentrations of reactive species by increasing the rate of ionization, and the time duration of exposure to plasma contributes to cancer treatment by increasing the number of species that come into contact with BHY cells [Figure 1]. As a matter of fact, increasing the reactive species endogenously over a certain threshold causes apoptosis, and hence regarding the anticancer effect of the plasma jet, it can be postulated that plasma therapy leads to the production of high levels of exogenous reactive species which, in turn, raises the endogenous concentrations through cellular uptake.

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Conflicts of interest

There are no conflicts of interest.

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  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]


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